H.C. Blue 1 is carcinogenic in the B6 mouse liver but not in the F344 rat, whereas H.C. Blue 2 is not carcinogenic in either species. The in vivo metabolism of the chemicals in the urine of both species, and the in vitro metabolism by hepatocytes from both species, have been analyzed. In vivo metabolism and hepatocyte metabolism gave similar HPLC profiles for each chemical. Rats and mice differed quantitatively in the H.C. Blue 1 metabolite profiles produced, which may contribute to the H.C. Blue 1's species specificity. The identity of specific metabolites are currently being identified. The adduction to DNA of H.C. Blue 1 to mouse and rat liver tissue had hepatocytes has been investigated. But studies have indicated an impurity(ies) may be responsible for adduct formation and therefore H.C. Blue 1 has been subjected to HPLC purification. From these studies, an impurity in H.C. Blue 1 responsible for mutagenicity and DNA adduction in Salmonella has been isolated. Seven DNA adducts have been identified in Salmonella. Purified H.C. Blue 1 is not mutagenic and does not form DNA adducts. Studies are continuing in an attempt to elucidate the basis of H.C. Blue 1's carcinogenic activity in the mouse and lack of carcinogenic activity in the rat.